Tissue-specific stem cells comprise only a small fraction of cells in adult organ but they provide the enormous proliferative reserve that allows for tissue repair and regeneration. These long-lived slow-cycling cells are postulated to be a prime target for the accumulated environmental injuries and genetic mutations that cause cancer, and are also thought to confer the unique therapy-resistant and hormone independent properties of difficult to treat malignancies such as breast and prostate cancer. Recent success in isolating mouse prostate epithelial cells has provided proof of concept for the existence of prostate stem cells. However, further progress is being hampered by rapid loss of their progenitor potential when cultured in standard monolayer conditions in vitro. This proposal describes a novel method to overcome that limitation by culturing prostate stem cells as anchorage-independent prostaspheres. Tissue specific stem cells exhibit a unique requirement for anchorage-independent growth in vitro. The recent development of anchorage-independent culture methods has allowed maintenance and proliferation of tissue specific stem cells from the brain and breast in vitro. Growth of these cells in the free-floating spheroids termed neurospheres and mammospheres, respectively, has been instrumental in moving the biology of tissue specific stem cells forward and in revealing the unique roles of Wnt and Hedgehog signaling in progenitor cell proliferation. In preliminary studies we have cultured prostate epithelial cells in anchorage- independent conditions and shown that the cells proliferating within the resulting "prostaspheres" exhibit increased expression of putative stem cell markers and exhibit activated Hh signaling. In this proposal, we will address the hypothesis that anchorage independent culture of prostate epithelial cells maintains a population of pleuripotent prostate stem cells. We will characterize the clonality, stem cell marker expression and regenerative potential of cells propagated in prostaspheres, rigorously test whether single cells derived through anchorage-independent culture exhibit the diverse differentiation potential of bona fide tissue specific stem cells, and then examine the role of Hh signaling, a critically important regulator of prostate development, in stem cell survival and proliferation. These goals will be achieved in three aims: 1) Characterize cells grown in prostaspheres, examine the clonality of individual spheres and correlate prostasphere-forming ability with expression of putative stem cell markers. 2) Use tissue recombination assays to test whether prostaspheres contain bona fide stem cells. 3) Examine the role of Hh signaling in prostasphere formation and stem cell proliferation. These studies will validate anchorage-independent culture as a method for growing prostate tissue-specific stem cells in vitro and overcome what is currently a huge obstacle to progress in the field. They will provide investigators with a simple method to expand, manipulate and study tissue-specific stem cells - an essential step in efforts to understand the origins of benign prostatic hyperplasia and prostate cancer. Additionally, our characterization of Hh signaling in stem cell proliferation is likely to provide new avenues to study of the role of Hh signaling in advanced prostate cancer and open novel routes for drug development. [unreadable] [unreadable] [unreadable]